We propose to apply the concepts learned from previous studies on the interrelationship between metebolism and transport in renal tubules to two broad areas: the effects of anoxia on proximal renal function and the metabolism of cultured renal proximal tubules. We will utilize suspensions of proximal tubules obtained from the rabbit and rat kidneys to study the tubular effects of anoxia independently from hemodynamic factors present during and after clamp-induced ischemia. Previous experiments in our laboratory subjecting the rabbit tubule suspension to anoxia and reoxygenation suggest that most of the cellular damage occurs during anoxia. The mechanism and sites of this damage will be investigated with a variety of interrelated approaches. The involvement of individual variables, such as calcium, ATP, and oxygen-derived free radicals will be evaluated. Cytosolic free calcium (Caf) will be measured using calcium-sensitive dyes and Caf will be modulated by use of calcium-selective ionophores and chelators. The effects of oxygen-free radicals will be tested during anoxia and reoxygenation by comparing rabbit and rat tubules, by addition of hypoxanthine and xanthine oxidase to the medium, and by the use of protective agents. Mitochondrial function will continue to be assessed independently from plasma membrane integrity. New techniques will be developed to measure membrane damage. Immunocytochemistry will be used to visualize the organization of cytoskeletal proteins within the microvilli and terminal web in association with the plasma membrane. Brush border membrane enzyme activity and plasma membrane phospholipid breakdown will also be measured during anoxia. This information is expected to have clinical relevance, since the added basic knowledge concerning the dysfunctions and protective mechanisms should suggest clinical interventions to ameliorate the dysfunctions. We have recently developed an improved rabbit proximal tubule suspension that has a normal viability of 1-3 days. This suspension will be used to study the conditions in the culture environment that cause a widely observed adaptation of cellular energy metabolism, such that oxidative metabolism decreases and glycotytic capacity is considerably enhanced. We will study the metabolic changes that occur as a function of partial oxygen pressure, metabolic substrates and mitogens, focusing on the conditions necessary for glycolytic enzyme induction. This knowledge will be used to attain our long term goal of developing the methodology to grow renal cells in monolayer culture that express normal oxidative metabolism.